PROJECT SUMMARY. Cyclophilin (Cyp) inhibitors (CypI) are clinically highly potent in HCV patients in pahse I and II studies. A recent phase I study showed that the daily administration of a CypI reduces viremia and rapidly increases plasma concentrations of IFN, 1 and 3, and 2'5'OAS-1. Changes in plasma concentrations for all markers were coincident with changes in plasma concentration of the CypI. These novel and attractive data revealed the first link between Cyps and the IFN response. We were able to partly reproduce the patient data in vitro. Specifically, CypI enhance secretion of IFN, and 2'5'OAS-1 from replicon cells. These data suggest that activation of the IFN response may represent a mechanism through which CypI exert their clinical antiviral activity. We investigated the possibility that the intracellular target for CypI, CypA, binds to components of the IFN response. Remarkably, we found that CypA binds to the IFN regulatory factor 9 (IRF9) via its isomerase pocket. CypI prevent IRF9-CypA interactions. CypA also binds to IRF3, 5 and 7, suggesting that CypA binds to all IRF members. Our work demonstrates for the first time that CypA binds specifically to a major class of elements of the IFN response - IRFs. It also reveals a novel opportunity to modulate the IFN response. In this application, we propose to extend this work by conducting a series of experiments aimed at dissecting the roles of CypA in the IFN response. This application should elucidate how CypA neutralization triggers IFN production and determine whether the CypI-mediated IFN production represents a new mechanism of antiviral action of CypI. This application should also determine what action or function CypA exerts on IRFs. Interestingly, our most recent work suggests that CypA, by interacting with IRFs, greatly impacts their degree of ubiquitination. We propose a new set of experiments aimed at identifying the link between the CypA-mediated IRF ubiquitination effect and the CypI-mediated activation of the IFN response. Our finding that CypI triggers IFN release from PBMCs isolated from HCV patients opens exciting new lines of enquiries. This ex vivo assay may allow us to determine whether CypI responders produce more IFN than CypI non-responders and whether the CypI response is genotype- or virus titer-specific. If this occurs, we then would use this information to develop an empiric pre-screen for subjects, who are likely to respond to a regimen comprising a CypI. We recently found that CypI administration to mice significantly inhibits the infectivity of a virus, which is unrelated to HCV, but which is also highly sensitive to IFN. Moreover, we found that the IFN plasma levels were superior in infected mice treated with CypI compared to those in untreated infected mice, suggesting that the CypI IFN induction is a more widely used mechanism. These remarkable preliminary finding opened new lines of exciting enquiries. The ultimate goal of this application is to improve our understanding of the role of Cyps in the IFN response to viral infection.